Sex steroid precursor alone or in combination with a selective estrogen receptor modulator and/or with estrogens and/or a type 5 cGMP phosphodiesterase inhibitor for the prevention and treatment of vaginal dryness and sexual dysfunction in postmenopausal women

ABSTRACT

Novel methods for treating or reducing the likelihood of acquiring vaginal dysfunctions, more particularly vaginal dryness and dyspareunia, leading to sexual dysfunction and low sexual desire and performance , in susceptible warm-blooded animals including humans involving administration of a sex steroid precursor. Further administration of estrogen or selective estrogen receptor modulator, particularly those selected from the group consisting of Raloxifene, Arzoxifene, Tamoxifen, Droloxifene, Toremifene, Iodoxifene, GW 5638, TSE-424, ERA-923, and lasofoxifene, and more particularly compounds having the general structure:  
                 
is specifically disclosed for the medical treatment and/or inhibition of development of some of these above-mentioned diseases. Pharmaceutical compositions for delivery of active ingredient(s) and kit(s) useful to the invention are also disclosed.

FIELD OF THE INVENTION

The present invention relates to a method for treating or reducing thelikelihood of acquiring problems affecting the layer lamina propria orthe layer muscularis of the vagina using sex steroid precursors alone orin a novel combination therapy on susceptible warm-blooded animals,including humans. In particular, the combinations include administeringa selective estrogen receptor modulator (SERM) and raising the patient'slevel of precursors of sex steroids, said precursor being selected fromthe group consisting of dehydroepiandrosterone (DHEA),dehydroepiandrosterone sulfate (DHEA-S), and androst-5-ene-3β,17β-diol(5-diol). Estrogens may also be administered to conteract the potentialeffects of some SERMs on hot flashes and other menopausal symptoms. Type5 cGMP phosphodiesterase inhibitor may also be administered to improvesexual activity. The invention also relates to kits and pharmaceuticalcompositions for practicing the foregoing combination.

In U.S. Pat. No. 5,843,932, was reported the effect on vaginal atrophyof one, three or six months treatment with DHEA administered at a doseof 30 mg twice daily in a solution of 50% ethanol-50% propylene glycolon an area of 2 cm² of the dorsal skin in ovariectomized rat.Histopathologic examination showed proliferation and murification of thevaginal epithelium and reversal of vaginal mucosal atrophy in the ratstreated with DHEA

The study performed with the rat when DHEA was applied on the dorsalskin for 1, 3 and 6 months examined only the effect on the vaginalepithelium (Sourla et al., 1998, J. Steroid Biochem Mol Biol., 66(3):137-149) and not on the two other layers, namely the lamina propria andthe muscularis. It was then observered that DHEA was about 10 times moreefficient on the vaginal epithelium when applied topically than whenadministered on the skin at a site distant from the vagina, thusrequiring systemic absorption to exert its action.

In a previous study on the effect of DHEA administered on the skin inpostmenopausal women for 12 months in a 10% DHEA cream, only theestrogenic activity of DHEA was evaluated (Labrie et al., 1997, J. Clin.Endocrinol. Metab., 82: 3498-3505). It was indicated (page 3500)“Vaginal cytology was examined as specific parameter of the estrogenicaction of DHEA”. This is due to the method of evaluation, namely vaginalsmear which is limited to the superficial and easily removed cells ofthe epithelium.

The present invention describes the effects of DHEA and other componentson the three layers of the vagina, namely the muscularis, the laminapropria and the epithelium with novel benefits at the three levels. Thebeneficial effects of DHEA on the lamina propria and muscularis arebelieved to be of major importance for the positive action of inhibitorsof type 5 cGMP phospho-diesterase, such as viagra and other compounds orprostaglandin E1.

BACKGROUND OF THE RELATED ART

Vaginal dryness affects about 50% of postmenopausal women at the age of50 to 60 years and 72% after 70 years (Rossin-Amar, 2000, Gynecol ObstetFertil, 28(3): 245-249). Of these women, about 80% experience urogenitaldisorders, especially vaginitis and dyspareunia (Pandit and Ouslander,1997, Am J Med Sci, 314(4): 228-31). Since these problems are believedto be at least partially related to a deprivation of sex steroids,appropriate local hormonal replacement therapy should be considered atmenopause. It has recently been recognized that postmenopausal women arenot only deprived of all ovarian estrogens but they are alsoprogressively deprived of the androgens originating from the peripheralintracrine transformation of dehydroepiandrosterone (DHEA) into bothandrogens and estrogens (Labrie et al., 1991, Mol Cell Endocrinol, 78:C113-C118; Labrie et al., 1995, Ann NY Acad Sci, 774: 16-28; Labrie etal., 2003, End Rev, 24(2): 152-182). In fact, serum DHEA and DHEA-Sprogressively decrease from the age of 30 to 40 years (Labrie et al.,2003, End Rev, 24(2): 152-182; Orentreich et al., 1984, J ClinEndocrinol Metab, 59: 551-555; Labrie et al., 1997, J Clin EndocrinolMetab, 82: 2396-2402). A series of studies indicate that low levels ofDHEA and DHEA-S are associated with a series of age-related morbidityand diseases (Labrie et al., 1997, J. Clin. Endocrinol. Metab., 82:3498-3505; Helzlsouer et al., 1992, Cancer Res, 52(1): 1-4; Szathmari etal., 1994, Osteoporos Int, 4(2): 84-88; Thoman and Weigle, 1989, AdvImmununol, 46: 221-261; Barrett-Connor et al., 1999, J Reprod Med,44(12): 1012-1020; Barrett-Connor et al., 1999, J Am Geriatr Soc, 47(6):685-691).

An efficient approach to alleviate vaginal dryness and other menopausalsymptoms is the use of hormone replacement therapy (HRT) (Greendale andJudd, 1993, J Am Geriatr Soc, 41(4): 426-436; Studd et al., 1980,Pasetto, Paleotti and Ambrus Eds, MT Press, Lancaster, p: 127-139).Recent clinical studies, however, have indicated that combiningestrogens and progestins increases the incidence of breast cancer with apotential negative impact on cardiovascular events (Colditz et al.,1995, N Engi J Med, 332: 1589-1593; Ross et al., 2000, J Natl CancerInst, 92(4): 328-332; Rossouw et al., 2002, JAMA, 288(3): 321-333).Meanwhile, there is an increasing interest in the potential of combinedestrogen-androgen replacement therapy (Rosenberg et al., 1997, J ReprodMed, 42(7): 394-404; Burd et al., 2001, Curr Women Health Rep,1(3):202-205), although the use of the estrogenic component is limitedby the potential complications mentioned above. Based upon recentadvances in our understanding of human sex steroid physiology,especially in postmenopausal women (Labrie et al., 1991, Mol CellEndocrinol, 78: C113-C118; Labrie et al., 2003, End Rev, 24(2):152-182), the use of DHEA becomes a possibility to providepostmenopausal women with the appropriate levels of androgens andestrogens synthesised in specific tissues by intracrine mechanisms, withno systemic effects (Labrie et al., 1997, J. Clin. Endocrinol. Metab.,82: 3498-3505: 16-28; Labrie et al., 2003, End Rev, 24(2): 152-182;Labrie, 2001, Ref Gyn Obstet, 8: 317-322; Lasco et al., 2002, 145:457-461). The restauration of androgen-sensitive elements of vaginalfunction should also help the action of inhibitors of type 5 cGMPphosphodiesterase or prostaglandin E1.

The selective estrogen receptor modulator (SERM) Acolbifene (EM-652) isa benzopyran derivative originally developed for the prevention andtreatment of breast cancer (Gauthier et al., 1997, J Med Chem, 40:2117-2122). Acolbifene is the compound having the highest affinity ofall known compounds for the ER (Gauthier et al., 1997, J Med Chem, 40:2117-2122; Labrie et al., 1999, J Steroid Biochem Mol Biol, 69 (1-6):51-84; Tremblay et al., 1997, Mol. Endocrinol., 11: 353-365) and itexerts its activity on both ERα and ERβ (Tremblay et al., 1998,Endocrinology, 139: 111-118). This compound displays a pure and highlypotent antiestrogenic activity in the mammary gland and endometriumwhile decreasing serum cholesterol and triglycerides and preventing boneloss, at least in the rat (Labrie et al., 1999, J Steroid Biochem MolBiol, 69 (1-6): 51-84). Moreover, it has been demonstrated that theadministration of DHEA, not only does not interfere, but does exert anadditive inhibitory effect with the pure antiestrogen Acolbifene onhuman breast tumour growth in the nude mouse (Dauvois et al., 1991,Cancer Res, 51: 3131-3135; Luo et al., 1997, Endocrinology, 138:4435-4444). Combined treatment of DHEA and Acolbifene has been proposedas a beneficial chemopreventive and therapeutic approach in breastcancer (Labrie, 2001, Ref Gynecol Obstet, 8: 317-322). In fact, theinhibitory effect of DHEA on the growth of human breast cancerxenografts in nude mice supports its use as hormone replacement therapy(Dauvois et al., 1991, Cancer Res, 51: 3131-3135; Couillard et al.,1998, J Natl Cancer Inst, 90: 772-778).

WO 99/63974 disclosed medical uses of a selective estrogen receptormodulator in combination with sex steroid precursors

SUMMARY OF THE INVENTION

It is accordingly an object of the present invention to provideeffective methods of treatment for vaginal problems, more particularlyvaginal dryness, dyspareunia, and sexual dysfunction which can lead todecrease in sexual desire and activity while minimizing undesirable sideeffects.

It is another object to provide methods of reducing the risk ofacquiring the above problems.

In one embodiment, the invention pertains to a method of treating orreducing the risk of acquiring vaginal dryness comprising increasinglevels of a sex steroid precursor selected from the group consisting ofdehydroepiandrosterone (DHEA), dehydroepiandrosterone-sulfate (DHEA-S)and androst-5-ene-3β,17β-diol (5-diol), in a subject or patient in needof said treatment or said steroid precursor, and further comprisingadministering to said patient a therapeutically effective amount of aselective estrogen receptor modulator (SERM) as part of a combinationtherapy.

In another embodiment, the invention includes additional administrationof estrogens to conteract the effects of SERMs on hot flashes and othermenopausal symptoms.

In another embodiment, the invention includes additional administrationof a Type 5 cGMP phosphodiesterase inhibitor or prostaglandin E1 toimprove sexual activity.

As used herein, a selective estrogen receptor modulator (SERM) is acompound that either directly or through its active metabolite functionsas an estrogen receptor antagonist (“antiestrogen”) in breast tissue,yet provides estrogenic or estrogen-like effect on bone tissue and onserum cholesterol levels (i.e. by reducing serum cholesterol).Non-steroidal compounds that function as estrogen receptor antagonistsin vitro or in human or rat breast tissue (especially if the compoundacts as an antiestrogen on human breast cancer cells) is likely tofunction as a SERM. Conversely, steroidal antiestrogens tend not tofunction as SERMs because they tend not to display any beneficial effecton serum cholesterol. Non-steroidal antiestrogens we have tested andfound to function as SERMs include EM-800, EM-01538, Raloxifene,Tamoxifen, Droloxifene, Toremifene, Idoxifene, TSE-424, ERA-923,Lasoxifene (CP 336156), Arzoxifene (LY 353 381) and GW-5638. We havetested the steroidal antiestrogen ICI 182,780 and found not to functionas SERM. SERMs, in accordance with the invention may be administered inthe same dosage as known in the art when these compounds are used asantiestrogens.

Without intending to be bound by theory, it is believed that SERMs, manyof which, preferably, have two aromatic rings linked by one to twocarbon atoms, are expected to interact with the estrogen receptor byvirtue of the foregoing portion of the molecule that is best recognizedby the receptor. Preferred SERMs have side chains which may selectivelycause antagonistic properties in breast tissue without havingsignificant antagonistic properties in other tissues. Thus, the SERMsmay desirably functions as antiestrogens in the breast whilesurprisingly and desirably functioning as estrogens (or providingestrogen-like activity) in bone and on the blood components (whereconcentrations of lipids and/or cholesterol are favorably affected). Thefavorable effect on cholesterol and/or lipids potentially translates toa favorable effect against atherosclerosis which is known to beadversely affected by improper levels of cholesterol and lipids.

In another embodiment, the invention includes method, pharmaceuticalcomposition and kit wherein the selective estrogen receptor modulatorhas a molecular formula with the following features:

-   a) two aromatic rings spaced by 1 to 2 intervening carbon atoms,    both aromatic rings being either unsubstituted or substituted by a    hydroxyl group or a group converted in vivo to hydroxyl;-   b) a side chain possessing an aromatic ring and a tertiary amine    function or salt thereof.    It is preferred that the side chain is selected from the group    consisting of:

It is also preferred that the two aromatic rings are both phenyl andthat the side chain possesses a moiety selected from the groupconsisting of a methine, a methylene, —CO, —O—, and —S—, an aromaticring, and a tertiary amine function or salt thereof.

In another embodiment, the selective estrogen receptor modulator isselected from the group consisting of a benzothiophene derivative,triphenylethylene derivative, indole derivative, benzopyran derivative,5,6,7,8-tetrahydronaphtalene and centchroman derivative.

In one embodiment, it is preferred that the selective estrogen receptormodulator is a benzothiophene derivative compound of the followingformula:

wherein R₁ and R₂ are independently selected from the group consistingof: hydrogen, hydroxyl, and a moiety converted in vivo in hydroxyl;

wherein R₃ and R₄ are either independently selected from the groupconsisting of: C1-C4 alkyl, or wherein R₃, R₄ and the nitrogen to whichthey are bound, together are any structure selected from the groupconsisting of pyrrolidino, dimethyl-1-pyrrolidino,methyl-1-pyrrolidinyl, piperidino, hexamethyleneimino and morpholino;

wherein A is selected from the group consisting of —CO—, —CHOH, and—CH₂—;

wherein B is selected from the group consisting of phenylene,pyridylidene, and -cycloC₄H₂N₂—.

Particularly, the selective estrogen receptor modulator is selected fromthe group consisting of Raloxifene, Arzoxifene (LY 353381) and LY335563.

In another embodiment, it is preferred that the selective estrogenreceptor modulator is a triphenylethylene derivative compound of thefollowing formula:

wherein D is —OCH₂CH₂N(R₃)R₄ or —CH═CH—COOH (R₃ and R₄ either beingindependently selected from the group consisting of C1-C4 alkyl, or R₃,R₄, and the nitrogen atom to which they are bound, together being a ringstructure selected from the group consisting of pyrrolidino,dimethyl-1-pyrrolidino, methyl-1-pyrrolidinyl, piperidino,hexamethyleneimino and morpholino);

wherein E and K are independently hydrogen or hydroxyl; wherein J ishydrogen or halogen.

Particularly, selective estrogen receptor modulator is Tamoxifen,OH-tamoxifen, Droloxifene, Toremifene, Iodoxifene, and GW 5638.

In another embodiment, it is preferred that the selective estrogenreceptor modulator is an indole derivative compound of the followingformula:

wherein D is selected from the groups consisting of —OCH₂CH₂N(R₇)R₈,—CH═CH—CO N(R₇)R₈, —CC—(CH₂)_(n)-N(R₇)R₈ (R₇ and R₈ either beingindependently selected from the group consisting of C₁-C₆ alkyl, or R₇,R₈ and the nitrogen atom to which they are bound, together being a ringstructure selected from the group consisting of pyrrolidino,dimethyl-1-pyrrolidino, methyl-1-pyrrolidinyl, piperidino,hexamethyleneimino, morpholino, ring);

wherein X is selected from the group consisting of: hydrogen, and C1-C6alkyl;

wherein R₁, R₂ R₃, R₄, R₅, and R₆ are independently selected from thegroup consisting of: hydrogen, hydroxyl, C₁-C₆ alkyl, and a moietyconverted in vivo in hydroxyl. Particularly, the selective estrogenreceptor modulator is TSE-424 and ERA-923.

In another embodiment, it is preferred that the selective estrogenreceptor modulator is a compound of the following formula:

wherein R₁ and R₂ are independently selected from the group consistingof: hydrogen, hydroxyl, and a moiety converted in vivo in hydroxyl;

wherein R₅ and R₆ are independently hydrogen or C₁-C₆ alkyl;

wherein D is —OCH₂CH₂N(R₃)R₄ (R₃ and R₄ either being independentlyselected from the group consisting of C₁-C₄ alkyl, or R₃, R₄ and thenitrogen atom to which they are bound, together being a ring structureselected from the group consisting of pyrrolidino,dimethyl-1-pyrrolidino, methyl-1-pyrrolidinyl, piperidino,hexamethyleneimino, morpholino).

Wherein X is selected from the group consisting of —O— and —CH₂—.

Particularly, the compound is selected from the group consisting of:(−)-cis-(5R,6S)-6-phenyl-5-[4-(2-pyrrolidin-1-ylethoxy)phenyl]-5,6,7,8-tetrahydronaphthalen-2-ol,D-(−)-tartrate salt (lasofoxifene) and(3,4-trans-2,2-dimethyl-3-phenyl-4-[4-(2-(2-(pyrrolidin-1-yl)ethoxy)phenyl]-7-methoxychroman).

In another embodiment, it is preferred that the selective estrogenreceptor modulator has the following formula:

wherein R₁ and R₂ are independently hydrogen, hydroxyl or a moiety whichis converted to hydroxyl in vivo;

-   wherein Z is a bivalent closing moiety, particularly, Z is selected    from the group consisting of —O—, —NH—, —S—, and —CH₂—;

wherein the R100 is a bivalent moiety which distances L from the B-ringby 4-10 intervening atoms;

wherein L is a bivalent or trivalent polar moiety selected from thegroup of —SO—, —CON—, —N<, and —SON<;

wherein G₁ is selected from the group consisting of hydrogen, a C₁ to C₅hydrocarbon or a bivalent moiety which joins G₂ and L to form a 5- to7-membered heterocyclic ring, and halo or unsaturated derivatives of theforegoing.

wherein G₂ is either absent or selected from the group consisting ofhydrogen, a C₁ to C₅ hydrocarbon or a bivalent moiety which joins G₁ andL to form a 5- to 7-membered heterocyclic ring, and halo or unsaturatedderivatives of the foregoing;

wherein G₃ is selected from the group consisting of hydrogen, methyl andethyl. More particularly, benzopyran derivatives of the followinggeneral structure are preferred:

wherein D is —OCH₂CH₂N(R₃)R₄ (R₃ and R₄ either being independentlyselected from the group consisting of C₁-C₄ alkyl, or R₃, R₄ and thenitrogen atom to which they are bound, together being a ring structureselected from the group consisting of pyrrolidino,dimethyl-1-pyrrolidino, methyl-1-pyrrolidinyl, piperidino,hexamethyleneimino, morpholino, ring).

wherein R₁ and R₂ are independently selected from the group consistingof: hydrogen, hydroxyl, and a moiety converted in vivo in hydroxyl.

It is also preferred that benzopyran derivatives are optically activecompounds having an absolute configuration S on carbon 2 orpharmaceutically acceptable salts thereof, said compounds having themolecular structure:

wherein R₁ and R₂ are independently selected from the group consistingof hydroxyl and a moiety convertible in vivo to hydroxyl;

wherein R³ is a species selected from the group consisting of saturated,unsaturated or substituted pyrrolidinyl, saturated, unsaturated orsubstituted piperidino, saturated, unsaturated or substitutedpiperidinyl, saturated, unsaturated or substituted morpholino,nitrogen-containing cyclic moiety, nitrogen-containing polycyclicmoiety, and NRaRb (Ra and Rb being independently hydrogen, straight orbranched C₁-C₆ alkyl, straight or branched C₂-C₆ alkenyl, and straightor branched C₂-C₆ alkynyl).

In one embodiment, the benzopyran derivative is a salt of an acidselected from the group consisting of acetic acid, adipic acid,benzenesulfonic acid, benzoic acid, camphorsulfonic acid, citric acid,fumaric acid, hydroiodic acid, hydrobromic acid, hydrochloric acid,hydrochlorothiazide acid, hydroxy-naphthoic acid, lactic acid, maleicacid, methanesulfonic acid, methylsulfuric acid,1,5-naphthalenedisulfonic acid, nitric acid, palmitic acid, pivalicacid, phosphoric acid, propionic acid, succinic acid, sulfuric acid,tartaric acid, terephthalic acid, p-toluenesulfonic acid, and valericacid.

The preferred selective estrogen receptor modulators are:

Raloxifene, Arzoxifene, Tamoxifen, Droloxifene, Toremifene, Iodoxifene,GW 5638, TSE-424, ERA-923, and lasofoxifene.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the Division along the longitudinal axis of the rat vaginainto seven cross-segments, from the external orifice (ostium)(segment 1) to the cervix level (segment 7) (portio vaginalis uteri).

FIG. 2 shows the vaginal epithelium histomorphology of segment 5 in thenine groups of rats. Bar in (J), 40 μm.

-   (A) As representative of the estrogenic effect, the stratified    squamous epithelium of cycling rats at estrous consists of four main    layers: one cell layer of stratum basale (b), 6 to 7 cell layers of    stratum spinosum (s) and a stratum granulosum of 5 to 6 layers (g)    overlaid by the stratum corneum (c) made of tightly packed flattened    cornified cells.-   (B) Cycling rats at proestrus which are under an    estrogenic-progestational influence, are used to illustrate    mucification. In most segments 2 to 7, one basal cell layer (b) was    overlaid by 5 to 6 cell layers of stratum spinosum (s), and a    stratum mucification (m) consisting of 3-4 layers of mucous cells.-   (C) In the OVX control, a basal cell layer (b) was overlaid by a    layer of atrophic cuboidal cells (a).-   (D) The vaginal epithelium of OVX rats treated with an daily oral    dose of Acolbifene, (2.5 mg/kg) shows atrophy, but with an outer    layer of low columnar mucous cells (m) overlying the basal cell    layer (b).-   (E) Vaginal epithelium of OVX rats treated with an daily oral dose    of Premarin (0.5 mg/kg). The OVX-induced atrophy was replaced by an    estrogenic pattern comparable to that found at estrus.-   (F) In OVX animals which received Premarin+Acolbifene, atrophy    predominated with a morphology similar to that of Acolbifene-treated    animals, although larger mucous cells were seen.-   (G) Following treatment of OVX animals with a once daily cutaneous    application of DHEA (80 mg/kg) on an area of 2×2 cm of the dorsal    skin, an hypertrophic epithelium made of 3-5 layers of mucous    cells (m) was seen overlying a basal layer (b). Several    invaginations characterized this epithelium (arrow).-   (H) In most areas of the vaginal epithelium of    DHEA+Acolbifene-treated animals, a layer of mucous cells (m) rested    on a basal cell layer (b), while in some areas, many layers of    mucous cells overlaid the basal cell layer. Several invaginations    characterized this epithelium (arrows).-   (I) Treatment with DHEA+Premarin led to a mixed epithelium composed    of a three to seven cell layer-thick stratified squamous    epithelium (s) overlaid by 3-5 layers of mucous cells (m) in 3    animals. In 2 animals, areas of stratified squamous epithelium were    predominant (insert). Bar in insert, 30 μm.-   (J) When DHEA, Premarin and Acolbifene were combined, the epithelium    was similar to that of the DHEA+Acolbifene group.

FIG. 3 shows the vaginal mucosa of OVX animals showing, in (A), severeinflammatory changes characterized by focal leukocyte infiltration withintraepithelial microabscess (M) and, in (B), focal erosion (E)characterized by reduced epithelial thickness and ulceration (U)visualized as a complete diseappearance of the epithelium.

FIG. 4 To illustrate the estrogen-like effect of Acolbifene on thevaginal epithelium lining segment 1 (external opening), comparisons withrelevant groups are made. In intact rats at estrus (A) and inPremarin-treated (D) animals, the epithelium is 10-15 layer-thick andkeratinized. In OVX controls (B), the epithelial thickness is reduced to4-6 layers and is generally not keratinized (see the absence of stratumgranulosum) while in Acolbifene-treated animals (C), the thickness isrestored to 9-11 layers and keratinized.

FIG. 5 shows the thickness (μm) of the three different vaginalcompartments at the level of the fifth segment of the rat vagina: a)epithelium, b) lamina propria, c) muscularis and d) total thickness,after 36 weeks treatment of OVX animals with DHEA, Premarin andAcolbifene, alone or in combination. Intact animals at estrus andproestrus are added as reference controls. Groups sharing the sameletter are not statistically different at p<0.05.

FIG. 6 shows the compactness of collagen fibers of the lamina propria isillustrated in segment 5 as low (A), moderate (B) or high, (C).

FIG. 7 shows microphotographs of the three vaginal compartments: (E)epithelium, (L) lamina propria and (M) muscularis, at the level of thefifth segment of the rat vagina with emphasis on the relative muscularisthickness in the different groups. Separation of the 3 vaginal walllayers with bars is indicated to best estimate the thicknessdistribution between the different groups. A) Intact, B) OVX, and OVXtreated with C) Acolbifene, D) Premarin, E) Premarin+Acolbifene, F)DHEA, G) DHEA+Acolbifene, H) DHEA+Premarin, I) DHEA+Premarin+Acolbifene.

FIG. 8 shows the vaginal weight measured 36 weeks after OVX andtreatment of OVX animals with Acolbifene, Premarin and DHEA alone or incombination. Intact animals are added as controls.

FIG. 9 shows the Immunohistochemical localization of AR, ERα and PR inthe (E) epithelium, (L) lamina propria and (M) muscularis at the levelof the fifth vaginal segment of the different groups. The bars indicatethe separation between the three vaginal compartments.

FIGS. 10 and 11 show the comparison between different SERMs: vaginalepithelial morphology, in rats.

FIG. 12 shows the picture of a microscope slide to display thepositioning of the 8 vaginal segments (the whole upper row and thesection at the right-down corner), followed by the 4 uterine segments (1segment of uterine neck and 3 segments of the two uterine horns—2sections per horn) in a sequence corresponding to their originalanatomical position. Segment 8 includes the lateral vaginal foldsneighbouring the beginning of the uterine cervix.

FIG. 13 shows the effect intact rat

At estrus (left column), the vaginal epithelium is 12-18 layer-thick insegment 4 and 12-15 layer-thick in segment 6.

At metestrus (right column), the vaginal epithelium displays 6 to 10cell layers in segment 4, and 5 to 7 layers in segment 6.

FIG. 14 shows the effect OVARIECTOMIZED UNTOUCHED Rats. In segment 4,the vagina is lined by a squamous stratified epithelium of 2 to 6 celllayers. In segment 6, complete atrophy is observed, with flat 2 to 3cell layers. Some rare foci of small cylindrical mucous cells could beseen throughout the whole vagina.

FIG. 15 shows the effect of placebo/suppository. In comparison with theOVX untouched group, the vaginal squamous stratified epithelium of theplacebo group was slightly thicker, with segment 4 displaying 4 to 8cell layers. In segment 6, the same morphology as that of OVX untouchedgroup was observed, with 2-3 epithelial cell layers. Foci of smallcylindrical mucous cells could be found throughout the vagina, slightlymore when compared with the OVX untouched animals.

FIG. 16 shows the effect of DHEA 0.33 mg/suppository. In segment 4, thevaginal epithelium is stratified squamous with 3 to 7 cell layers. About20 to 60% of the vaginal lining consists of well-aligned mucous cells inalternance with 15 to 50% of hypertrophied mucous cells, overlying oneor more squamous stratified epithelium layers, and with non-mucifiedareas. In segment 6, the epithelial thickness is reduced to almost thethickness found in OVX untouched animals: 2-5 cell layers overlaid bycylindrical mucous cells. A large variation in the occurrence of mucouscells was observed, these cells covering from 5 to 75% of the vaginalsurface, in alternance with non-mucified areas.

FIG. 17 shows the effect of DHEA 0.66 mg/suppository. In segment 4, thestratified squamous epithelium is 3 to 6 cell layer-thick. About 5 to20% of the vaginal lining consists of well-aligned mucous cells inalternance with 10 to 75% of hypertrophied mucous cells, overlying oneor more squamous stratified epithelium layers. Non-mucified epithelialareas are also present. In segment 6, the epithelial thickness isreduced and consists of a squamous stratified epithelium of 2 to 4 celllayers, surmounted by a layer of well-aligned mucous cells. Mucifiedareas varied from 20 to 80% of the vaginal surface, in alternance withnon-mucified areas.

FIG. 18 shows the effect of DHEA 1 mg/suppository. In segment 4, thestratified squamous epithelium displays 3 to 9 cell layers.Hypertrophied mucous cells overlying 3 to 4 squamous cell layers cover50 to 70% of the vaginal surface, while well-aligned mucous cells line 5to 30% of the vaginal surface. Non-mucified areas are interspersedbetween mucified cells. In segment 6, the epithelial thickness is alsoreduced and consists of 2 to 4 cell layers. Hypertrophied mucous cellsoverlying 1 to 3 squamous cell layers cover about 30 to 100% of thevaginal surface while well-aligned mucous cells cover 0 to 70%. Fewinterspersed non-mucified areas are also observed.

FIG. 19 is a graph of the effect on body weight of 14-day treatment withDHEA (suppository) applied intravaginally in OVX rats. Compared withplacebo group, the body weight of the groups given DHEA at doses of 0.33mg and 0.66 mg slightly decreased, while the decrease was morepronounced in the 1 mg group.

FIG. 20 is a graph of the serum concentration of DHEA followingintravaginal application of 0.33 mg, 0.66 mg or 1 mg to OVX rats. Areaunder the curve (AUC) values seven hours following DHEA treatment were32±4 ng.h/ml, 50±7 ng.h/ml and 131±23 ng.h/ml when DHEA was given at thedoses of 0.33 mg, 0.66 mg or 1 mg, respectively.

DETAILED DESCRIPTION OF THE INVENTION

After cessation of estrogen secretion by the ovaries at menopause,practically all androgens and estrogens are synthesized in peripheraltarget tissues by intracrine mechanisms from dehydroepiandrosterone(DHEA) of adrenal origin. In fact, in the absence of ovarian estrogens,the gradual decrease in serum DHEA is likely to play an important rolein the vaginal dryness, inflammation, dyspareunia and sexual dysfunctionfrequently associated with menopause. In order to assess the specificestrogenic and/or androgenic effects of a potential novel hormonereplacement therapy that could, among other beneficial effects,alleviate vaginal dryness, we have examined the morphology of the ratvagina eight months after ovariectomy (OVX) and treatment of OVX animalswith DHEA, conjugated estrogens (Premarin) and the selective estrogenreceptor modulator Acolbifene, administered alone or in combination. Inintact animals at estrus and in OVX rats treated with Premarin, thetypical vaginal estrogenic pattern is a keratinized stratified squamousepithelium. OVX led to a general atrophy associated with inflammatorychanges while Acolbifene reduced the inflammation incidence andincreased the number and size of mucous cells in the vaginal epithelium.At the doses used, Premarin completely reversed the OVX-inducedepithelial atrophy, while DHEA partially reversed the atrophy. In fact,the vaginal epithelium of OVX animals treated with DHEA becamehyperplastic with 3-5 layers of columnar mucous and goblet cells typicalof an androgenic effect. The addition of Premarin to DHEA led to anepithelium thicker than in intact animals. Moreover, compactness of thecollagen fibers in the lamina propria was increased by DHEA. On theother hand, treatment with Acolbifene alone showed a tendency for anincrease in the lamina propria thickness, which reached intact valueswhen combined with DHEA.

After OVX, the vaginal muscular layer was decreased by 46%, an effectwhich was 41% and 100% reversed by DHEA and Premarin, respectively. Onthe other hand, the 50% decrease in total vaginal wall thicknessfollowing OVX was 42% and 93% reversed by DHEA and Premarin,respectively, while the combination of DHEA and Acolbifene reversedtotal vaginal wall thickness to a value not significantly different fromintact control animals.

Immunohistochemistry revealed strong androgen receptor (AR) labeling inall DHEA-treated groups. On the other hand, estrogen receptor alpha(ERα) and progesterone receptor (PR) labeling were not detected in anyof the Acolbifene-treated groups.

In conclusion, treatment with DHEA or the combination of DHEA andAcolbifene partially or completely prevents the OVX-induced atrophicchanges observed in various layers of the vaginal wall through apredominant androgenic effect, as revealed by epithelial mucificationand AR up-regulation. The present data also show particularlyinteresting effects of DHEA on the three layers of the vaginal wall,namely a highly mucified epithelium, an increased compactness of thecollagen fibers in the lamina propria as well as an increase of themuscularis thickness. DHEA thus exerts both androgenic and estrogeniceffects on the vaginal mucosa, thus providing a more physiologicalreplacement therapy. While each of the examined compounds has potentialbeneficial effects on vaginal function, DHEA alone or in combinationcould well optimally relieve vaginal dryness and restore global vaginalphysiology and help correcting sexual dysfunction associated withmenopause. DHEA and other components could be administered locally orsystemically.

A selective estrogen receptor modulator of the invention has a molecularformula with the following features: a) two aromatic rings spaced by 1to 2 intervening carbon atoms, both aromatic rings being eitherunsubstituted or substituted by a hydroxyl group or a group converted invivo to hydroxyl; and b) a side chain possessing an aromatic ring and atertiary amine function or salt thereof.

One preferred SERM of the invention is EM-800 reported in

Another preferred SERM of the invention is acolbifene (EM-1538, alsocalled EM-652.HCl) reported in U.S. Pat. No. 6,710,059 B1

is the hydrochloride salt of the potent antiestrogen EM-652. Compared toEM-800, EM-1538 is a safter, simpler, and easier salt to synthesize. Inadministering either EM-800 or EM-1538, it is believed to result in thesame active compound in vivo.

Another prefered SERM is Lasoxifene (CP-336,156;(−)-cis-(5R,6S)-6-phenyl-5-[4-(2-pyrrolidin-1-ylethoxy)phenyl]-5,6,7,8-tetrahydronaphthalen-2-ol,D-(−)-tartrate salt) (available from Pfizer Inc., USA).

Other preferred SERMs of the invention include Tamoxifen((Z)-2-[4-(1,2-diphenyl-1-butenyl)]-N,N-dimethylethanamine) (availablefrom Zeneca, UK), Toremifene (available from Orion-Farmos Pharmaceutica,Finland, or Schering-Plough), Droloxifene, and Raloxifene (Eli Lilly andCo., USA), Arzoxifene (LY 335563) and LY 353381 (Eli Lilly and Co.,USA), Ospemifene (FC 1271) (available from Orion-Farmos Pharmaceutica,Finland), Iodoxifene (SmithKline Beecham, USA), Levormeloxifene(3,4-trans-2,2-dimethyl-3-phenyl-4-[4-(2-(2-(pyrrolidin-1-yl)ethoxy)phenyl]-7-methoxychroman)(Novo Nordisk, A/S, Denmark) which is disclosed in Shalmi et al. WO97/25034, WO 97/25035, WO 97/25037,WO 97/25038; and Korsgaard et al. WO97/25036), GW-5638 (described by Willson et al., 1997, Endocrinol,138(9): 3901-3911), SERM 3339 in development by Aventis (France) andindole derivatives (disclosed by Miller et al. EP 0802183A1) and TSE-424and ERA-923 developed by Wyeth Ayers (USA) and disclosed in JP10036347(American home products corporation) and nonsteroidal estrogenderivatives described in WO 97/32837.

Any SERM used as required for efficacy, as recommended by themanufacturer, can be used. Appropriate dosages are known in the art. Anyother non steroidal antiestrogen commercially available can be usedaccording to the invention. Any compound having activity similar toSERMs (example: Raloxifene can be used).

SERMs administered in accordance with the invention are preferablyadministered in a dosage range between 0.01 to 10 mg/kg of body weightper day (preferably 0.05 to 1.0 mg/kg), with 10 mg per day, especially20 mg per day, in two equally divided doses being preferred for a personof average body weight when orally administered, or in a dosage rangebetween 0.003 to 3.0 mg/kg of body weight per day (preferably 0.015 to0.3 mg/ml), with 1.5 mg per day, especially 3.0 mg per day, in twoequally divided doses being preferred for a person of average bodyweight when parentally administered (i.e. intramuscular, subcutaneous orpercutaneous administration). Preferably the SERMs are administeredtogether with a pharmaceutically acceptable diluent or carrier asdescribed below.

Preferred type 5 cGMPphosphodiesterase inhibitor are Sildenafil marketedunder the Tradename “Viagra” by Pfizer USA, Tadalafil marketed under theTradename “Cialis” by Eli Lilly USA, vardenafil marketed under theTradename “Levitra” by Bayer (Germany). It is also preferred type 5cGMPphosphodiesterase inhibitor which are presently in development:DA-8159 by Dong-A Pharm Tech (South Korean), EMR-62203 by Merck(Germany), TA-1790 by Tanabe Seiyaku (Japan), SCH-446132 bySchering-Plough (U.S.A.), and UK-371800 by Pfizer (U.S.A.).

One preferred prostaglandin is alprostadil marketed under the Tradename“Alprox-TD” by NexMed (USA).

With respect to all of the dosages recommended herein, the attendingclinician should monitor individual patient response and adjust dosageaccordingly.

EXAMPLE OF EFFICACY OF THE INVENTION Example 1

Materials and Methods

Animals and Treatments

Ten to twelve week-old female Sprague-Dawley rats (Crl:CD®(SD)BrVAF/Plus™) (Charles River Laboratory, St-Constant, Canada) weighingapproximately 220-270 g at start of the experiment were used. Theanimals were acclimatized to the environmental conditions (temperature:22±3° C.; humidity: 50±20%; 12-h light-12-h dark cycles, lights on at07:15 h) for at least one week before starting the experiment. Theanimals were housed individually and were allowed free access to waterand rodent food (Lab Diet 5002, Ralston Purina, St-Louis, Mo.). Theexperiment was conducted in accordance with the CCAC Guide for Care andUse of Experimental Animals in an animal facility approved by theCanadian Council on Animal Care (CCAC) and the Association forAssessment and Accreditation of Laboratory Animal Care (AAALAC).

A total of 126 female rats were randomly distributed into 9 groups of 14animals each as follows: 1) Intact control; 2) Ovariectomized control(OVX); 3) OVX+Acolbifene (2.5 mg/kg); 4) OVX+Premarin (0.5 mg/kg); 5)OVX+Premarin+Acolbifene; 6) OVX+DHEA (80 mg/kg); 7) OVX+DHEA+Acolbifene;8) OVX+DHEA+Premarin; 9) OVX+DHEA+Acolbifene+Premarin. On the first dayof the study, the animals of all groups (except group one) werebilaterally ovariectomized (OVX) under isoflurane-induced anesthesia.Premarin and Acolbifene were administered by oral gavage (0.5 ml/rat) assuspensions in 0.4% methylcellulose while DHEA in 50% ethanol-50%propylene glycol (0.5 ml/rat), was topically applied on a shaved area of2×2 cm of the dorsal skin. Dosage selection for Premarin corresponds tothe minimal dose sufficient to reverse OVX-induced uterine atrophy,while Acolbifene was administered at a dose sufficient to cause uterineatrophy similar to OVX after its administration to Premarin-treated OVXanimals. The dose of DHEA used gave DHEA blood levels at 70-100 nmol/L.Treatments were initiated on day 2 of the study and the compounds wereadministered once daily for 36 weeks. Animals from the intact and OVXcontrol groups received the vehicle alone.

Twenty-four hours after the last dosing, overnight fasted animals weresacrificed under isoflurane anesthesia by exsanguination at theabdominal aorta (9 animals per group) or by intracardiac perfusion with10% neutral buffered formalin (5 animals per group). Vaginae fromnon-perfused animals were collected and weighed, while the vaginaecollected from perfused animals were marked with black ink on theventral side, and then trimmed as described below.

Histological Procedures

The entire vagina of each perfused animal was post-fixed in 10% neutralbuffered formalin. Each vagina was then divided into seven equalcross-segments as illustrated in FIG. 1, routinely processed andembedded altogether in the same paraffin block. Within the paraffinblock, the seven vaginal cylindrical segments were positioned in asequence corresponding to their original anatomical position andoriented perpendicular to the surface of the block, thus allowing thesegments to be cut in cross-sections. For each animal, a 4 μm-thickparaffin section was cut and stained with haematoxylin-eosin formorphological examination.

Histomorphometry

Measurements of the different vaginal layers were performed on the fifthsegment (FIG. 1), which is approximately halfway between the middleregion and the portio vaginalis uteri (segment 7). This fifth segmentwas found to display a representative epithelial surface and asufficient thickness of smooth muscle. Images were captured with aDC-330 3CCD color camera (Dage-MTI, Michigan City, Ind., USA) andquantified using Image-Pro Plus 3.0 software (Media Cybernetics, SilverSpring, Md., USA). Thus, using a ×5 objective (Leica Microsystems,Willowdale, Ont., Canada), 3 to 4 thickness measurements per layer wereobtained from representative areas of the epithelium and muscularis, andfor the three vaginal layers together. The total vaginal thickness, thethickness of the epithelium and the thickness of the muscularis werethus measured. The thickness of the lamina propria was obtained bysubtracting the thickness of the epithelium and muscularis from totalvaginal thickness.

Immunohistochemistry

Immunostaining was performed using Zymed SP kits (San Francisco,Calif.). Paraffin sections (4 μm) were deparaffinized in toluene andrehydrated through ethanol. Endogenous peroxidase activity waseliminated by preincubation with 3% H₂O₂ in methanol for 30 min. Amicrowave retrieval technique using citrate buffer (Tacha and Chen,1994) was applied. After cooling the slides, non-specific binding wasblocked using 10% goat serum for 20 min. Sections were then incubatedfor 1.5 h at room temperature with ERα (AB-1, Calbiochem, California),AR (N-20, Santa Cruz Biotechnology, California) or PR (Ab-4, NeoMarkers,California) antibodies, at 1:200, 1:250 and 1:250 respectively. Afterwashing in PBS buffer, sections were incubated with biotinylatedanti-rabbit secondary antibody for 10 min and thereafter withstreptavidin-peroxidase for another 10 min. Diaminobenzidine was used asthe chromogen to visualize the biotin/streptavidin-peroxidase complex,under microscope monitoring. Counterstaining was performed using #2Gill's hematoxylin for 30 sec. For controls, immunoabsorption with anexcess of the peptide used to raise the antibody, or substitution withnon immune rabbit IgG, was performed. Semiquantitative evaluation of thenumber and intensity of immunostained nuclei was performed as indicatedin Table 2. TABLE 1 Histological evaluation of the epithelium, laminapropria and muscularis in the seven rat vaginal segments. VaginalSegment Segment Segment Segment Segment Segment Segment Group walls 1 23 4 5 6 7 INTACT estrus E KS KS KS KS KS KS KS L T¹-L² t-L t-L t-M M-MM-M M-M M s s t M T T T proestrus E KS MSM - KS MSM MSM MSM - KS MSM -KS MSM - KS L T-M t-M t-M t-M t-M M-M M-M M s s t M M T T OVX Control EA A A A A A A L t-H t-M t-H t-H t-H M-M M-H M s s t t t t M Acolbifene EKS SCM - A SCM - A SCM - A SCM - A SCM - A SCM - A L M-M t-M t-H t-H t-Ht-H M-H M s s s s t t t Premarin E KS KS - MSM KS - MSM KS - MSM KS -MSM KS KS L M-L t-M t-M M-M M-M M-H T-H M s s t M T T T Premarin + E KSA - SCM SCM - A SCM - A SCM - A SCM - A SCM - A Acolbifene L M-M t-M t-Ht-H M-M M-M M-M M s s t t t t M DHEA E KS - LHM LHM LHM LHM LHM LHM -SCM LHM - SCM L M-H t-H t-H t-H t-H M-H M-H M s s s t M M M DHEA + EKS-LHM- SCM - LHM SCM - LHM SCM - LHM SCM - LHM SCM - LHM LHM - SCMAcolbifene SCM L M-M t-M t-H t-H M-H M-H M-H M s s s t t M M DHEA + E KSMSM - KS MSM - KS MSM - KS MSM - KS KS KS Premarin L M-M t-L t-M t-M M-HM-M M-H M s s t M T T T DHEA + E KS SCM - LHM-KS SCM - LHM SCM - LHMSCM - LHM SCM - LHM LHM - SCM Premarin + L M-M M-H t-H M-H t-H M-H M-MAcolbifene M s s s t M M ME = epithelium morphology: KS (keratinized stratified squamous), LHM(large hypertrophied mucous cells), SCM (small aligned columnar orcuboidal mucous cells), MSM (mixed stratified squamous overlaid bymucous cells), A (atrophy: small cuboidal cells).Two different abreviations for a given segment indicate two differentpatterns and that the first one is the predominant.¹L = lamina propria thickness: T (thick), MT (moderately thick), t(thin);²compactness of collagen fibers: H (high), M (moderate), L (low);M = muscularis thickness: T (thick), MT (moderately thick), t (thin), s(scarce).

TABLE 2 Semiquantitative evaluation of the number and intensity ofimmunostained nuclei for AR, ER alpha and PR in the epithelium, laminapropria and muscularis in the fifth segment of the rat vagina. Sexsteroid receptor AR ERα PR Vaginal layer Group E L M E L M E L M IntactEstrus 2 2 1 1 1 3 2 3 3 Reference ++ +++ + + + + +++ +++ +++ of low ERαand high PR Diestrus 3 3 3 3 1 1 0 2 2 Reference +++ +++ +++ +++ ++ + ++++ of high ERα and low PR OVX control 1 1 1 2 1 1 0 0 0 ++ +++ +++ +++++ +++ Acolbifene 2 1 1 0 0 0 0 0 0 ++ +++ +++ Premarin 2 2 1 2 3 3 2 23 ++ +++ + ++ ++ ++ ++ +++ +++ Premarin + 2 1 1 0 0 0 0 0 0 Acolbifene++ + + DHEA 3 3 3 2 2 3 0 0 1 +++ +++ +++ ++ +++ +++ ++ DHEA + 3 3 3 0 00 0 0 0 Acolbifene +++ +++ +++ DHEA + 2 3 3 2 2 3 2 2 3 Premarin +++ ++++++ + +++ +++ ++ +++ +++ DHEA + 3 3 3 0 0 0 0 0 0 Premarin + +++ +++ +++AcolbifeneAndrogen receptor (AR), estrogen receptor alpha (ERα), progesteronereceptor (PR), epithelium (E), lamina propria (L), muscularis (M).Numbers represent the semi-quantitative evaluation of labeled nuclei: 0= none, 1 = low, 2 = moderate, 3 = highLabeling intensity is indicated as low: +, moderate: ++ and high: +++Statistical Analysis

Data are presented as means±SEM of 8-9 animals per group for vaginalweight or 5 animals per group for vaginal layer thicknessdeterminations. Statistical significance was determined according to themultiple-range test of Duncan-Kramer (Kramer, 1956, Biometrics, 12:307-310).

Results

Morphology and Thickness of the Different Layers of Rat Vagina

To examine with precision and detail the three layers of the rat vaginalwall, namely the epithelium, lamina propria and muscularis, sevensegments obtained along the longitudinal axis (FIG. 1) were firstexamined. While important morphological differences were observedbetween the groups, in general, the differences were uniform in allanimals of the same group and segments 2, 3, 4, 5, 6 and 7 have shown asimilar epithelial morphology. The few exceptions observed will bementioned later. Segment 5 was thus used to illustrate the effect of thevarious treatments on the vaginal epithelium.

In the OVX group, the absence of ovarian stimulation led to an atrophyof the vaginal epithelium which characterized all segments (Table 1 andFIG. 2C) with the presence of some small mucous cell areas. Mostimportantly, moderate to severe inflammation with many foci ofintraepithelial microabcesses (small areas of agglomerated leucocyteswithin the epithelium) were a frequent finding. These changes werefrequently accompanied by focal erosion (zones where the epitheliallayer partially disappears) and ulceration (complete disappearance ofthe epithelial layer) (FIG. 3).

In OVX animals treated with Acolbifene, segment 1 showed a keratinizedstratified squamous epithelium similar to that of the intact group,except that it included 9-11 cell layers compared to the 10-15 layersfound in animals at estrus or when OVX animals received Premarin (FIG.4). The epithelial thickness of Acolbifene-treated animals in segment 1was thus higher than that of OVX rats, which comprised only 4-6 celllayers. In the other segments (Table 1), the basal layer was covered bya layer of low columnar mucous cells (FIG. 2D), which were moredeveloped than in OVX animals. In the OVX+Acolbifene group, only threeout of five animals showed signs of minimal inflammation while moderateto severe inflammatory changes were general findings in all OVX animals.

In all groups, segment 1 showed a comparable stratified squamousepithelium, except in the animals of the OVX group where atrophy was apredominant characteristic (FIG. 4). Moreover, in the OVX+DHEA andOVX+DHEA+Acolbifene groups, mucous cells frequently accompanied thedominant stratified squamous epithelium of segment 1 (not shown).

The seven segments of the vaginal epithelium of OVX animals whichreceived DHEA were composed of large multilayered columnar mucous cellswith distended cytoplasmic vacuoles, a feature typical of an androgeniceffect (Table 1, FIG. 2G). Several large invaginations characterized theepithelium after DHEA treatment. A similar epithelial morphology wasfound in all segments of the OVX+DHEA+Acolbifene group, except that thesize of the mucous cells and the number of their layers were decreased(Table 1, FIG. 2H). In the DHEA+PREMARIN-treated animals, a thickstratified epithelium of a “mixed” type composed of different ratios ofsquamous epithelium covered by layers of mucous cells (Table 1, FIG. 21)was observed from the second to the fifth segment, thus revealingcombined estrogenic and androgenic effects. In this group, three animalsdisplayed mucification over the above-mentioned segments, while in theother two animals, a stratified squamous epithelium was observed in allsegments (Table 1). When PREMARIN was combined with DHEA and Acolbifene,the epithelium was similar to that of the DHEA+Acolbifene group, thusindicating a blockade of the estrogen-induced squamous cellproliferation by Acolbifene (Table 1 and FIG. 2J).

The administration of DHEA to OVX animals increased thickness of thevaginal epithelium to 38±4 μm, while the addition of Acolbifene to DHEAdid not change the epithelial thickness which remained at 37±5 μm. WhenPremarin and DHEA were coadministered to OVX animals, a high thicknessof 84±6 μm was observed, a value not significantly different from thatof the group of animals which received Premarin alone. Finally, thecombination of Premarin, DHEA and Acolbifene resulted in an epitheliumof 32±3 μm-thick, a value similar to that of the groups which receivedDHEA alone or DHEA combined with Acolbifene.

Lamina Propria

The degree of compactness of collagen fibers in the vagina wascategorized as low, moderate and high (FIG. 6), as observed in the areaproximal to the epithelium. <<Low>> and <<moderate compactness>> wereassociated with the presence of coarse collagen fibers loosely or lessloosely aggregated together, respectively, while <<high compactness>>was the term used to describe tightly packed fine collagen fibers,displaying a smooth, textured appearance. In all the animals, there wererelatively few fibrocytes in proportion to the amount of collagen, andthey appeared predominantly flattened and shrunken.

Careful examination of each animal (Table 1) reveals that, in general,the compactness of the collagen fibers in segment 1 is moderate (exceptlow in rats at estrus and in Premarin-treated OVX animals, or high inOVX and DHEA-treated OVX rats), while increasing in compactness insegments 2 and 3 to reach a plateau that generally remains constantuntil segment 7. Thus, along the longitudinal axis of the vagina inintact rats at estrus, compactness of the collagen fibers was low insegments 1 to 3 and moderate in segments 4 to 7. Atrophy was oftenassociated with increased compactness of collagen fibers in the OVX andOVX+Acolbifene groups (Table 1). In Premarin-treated OVX animals,compactness in segment 1 was low and moderate in segments 2 and 3, whilein the other segments, compactness of the collagen fibers increasedgradually to become high in segments 6 and 7.

Visual light microscope examination of the lamina propria thicknessalong the vagina generally revealed that is was moderately thick insegment 1 while thinner in segments 2 to 4. In segments 5 to 7, thethickness increased progressively to a level similar to segment 1. Thecorresponding mean values of lamina propria thickness measured insegment 5 (FIG. 5B) indicate that OVX led to a significant decrease inlamina propria thickness (76±2 μm versus 135±28 μm in the intact group)with a non significant decrease induced by Acolbifene (60±8 μm). Theincrease observed after Premarin or Premarin+Acolbifene administrationin OVX animals remained below the intact group (100±9 μm and 90±3 μm,respectively versus 135±28 μm). The administration of DHEA led also to astatistically non significant increase in lamina propria thickness(96±20 μm), and the addition of Acolbifene led to further increasedthickness (136±17 μm), reaching that of intact animals. Treatment of OVXanimals with Premarin+DHEA significantly increased the thickness,(144±14 μm) when compared to DHEA alone. Finally, the animals treatedwith Premarin+DHEA+Acolbifene displayed a thickness (99±9 μm) similar tothat of the group treated with DHEA alone, and lower than that of thePremarin+DHEA group, although the difference was not statisticallysignificant.

DHEA induced a moderate increase in muscularis thickness (50±2 μm),which was slightly decreased by the addition of Acolbifene (41±3 μm).Finally, treatment with Premarin and DHEA combined resulted in a notablethickness increase (62±3 μm), when compared to animals treated with DHEAalone. When Acolbifene was added to Premarin and DHEA, muscularisthickness decreased (46±5 μg) to a value comparable to DHEA andAcolbifene.

When total vaginal wall thickness was measured (FIG. 5D), the outer thinlayer of connective tissue composing the adventitia was not included.OVX led to a marked (51%) vaginal wall atrophy (128±3 μm, versus 262±39μm in the intact) and the addition of Acolbifene to OVX had no effect(108±8 μm) (FIGS. 7 A, B and C). Premarin treatment maintained the totalthickness to a value (253±10 μm) similar to that of intact animals whilethe addition of Acolbifene to Premarin reversed the effect of Premarin(150±4 μm) (FIGS. 7D and E, respectively). Total vaginal thicknessachieved by DHEA treatment (184±21 μm) was about 25% lower than that ofthe Premarin alone-treated group (FIG. 7F). On the other hand, theaddition of Acolbifene to DHEA led to a non significant thicknessincrease (213±20 μm), which became non significantly different from theintact group (FIG. 7G). Finally, coadministration of Premarin and DHEAto OVX animals markedly increased the thickness (290±13 μm) to a valuesimilar to that of intact animals (FIG. 7H). The addition of Acolbifeneto DHEA and Premarin reversed the effect to a value (176±11 μm) notsignificatively different from DHEA alone (FIG. 7I).

Vaginal Weight

After eight months of treatment, the changes observed in vaginal weightbetween the different groups (FIG. 8) generally follow theabove-described morphological observations. Indeed, vaginal weight afterOVX decreased by about 50% (101±5 mg) compared to the intact group(205±11 mg) while treatment of OVX animals with Acolbifene alone had noeffect on vaginal weight. On the other hand, administration of Premarinled to a vaginal weight increase that did not yet reach the value ofintact animals (170±9 mg) while the addition of Acolbifene to Premarinreversed the estrogen-induced weight gain to a value similar to that ofOVX animals (96±4 mg). Conversely, when DHEA was given to OVX animals,vaginal weight increased to a value (171±12 mg), similar to that of thePremarin-treated group, and the combination of Acolbifene and DHEAresulted in a decrease in weight (135±9 mg), which remained above thatof the OVX group. Finally, coadministration of Premarin and DHEA in OVXanimals resulted in a vaginal weight gain (179±10 mg) reaching a valuesimilar to those of the OVX+DHEA and OVX+Premarin groups. The additionof Acolbifene to this combination had no significant effect (194±12 mg).

DHEA treatment of OVX animals led to the strong labeling or AR of manynuclei in the three vaginal layers, the same pattern being found whenthe combinations of DHEA+Acolbifene and DHEA+Premarin were used, withthe exception of a smaller number of labeled nuclei in the superficiallayers of the epithelium in the latter group. The combination of DHEA,Premarin and Acolbifene also resulted in a strong staining of themajority of the nuclei in the three vaginal wall layers.

Discussion

Vaginal dryness or atrophic vaginitis, also referred to as urogenitalatrophy, with sexual dysfunction is a common problem in postmenopausalwomen (Notelovitz, 2000, Menopause, 7(3): 140-142). The most commonsymptoms are dryness, burning, pruritus, irritation and dyspareunia,thus leading to decreased libido and quality of life (Berman et al.,1999, Curr Opin Urol, 9(6): 563-568). Since estrogen loss is known to beinvolved, estrogen replacement therapy (ERT) and HRT are the treatmentsof choice. However, as new information on sex steroid physiology inwomen strongly suggests an important role of androgens (Labrie et al.,2003, End Rev, 24(2): 152-182), the present study compares the overallhormonal effects of an alternative to HRT or ERT, namely DHEA alone orin association with the pure antiestrogen Acolbifene and also withPremarin on rat vaginal morphology and sex steroid receptor expression.DHEA was administered percutaneously to avoid first pass through theliver (Labrie et al., 1996, Endocrinol, 150: S107-S118).

Finally, our results have shown a significant vaginal weight increasewith Premarin treatment, when compared to OVX animals, the value beingsimilar to that observed in DHEA-treated group. It is appropriate torecall that, although vaginal and uterine weight increases in rodentsare commonly used as measures of estrogenicity, these organs can alsorespond to progesterone and testosterone, among other compounds (Emmensand Martin, 1964, Dorfman Ed, Ed Academic Press NY: 1).

In the potential triple combination, the equine estrogen Premarin isaimed at acting in the brain to relieve the vasomotor symptoms. In fact,the benefits of co-administration of the pure selective antiestrogenAcolbifene with an estrogen in order to neutralize the unwantedperipheral effects of the estrogen have been well described by Labrie etal. (Labrie et al., 2003, Endocrinol, 144 (11): 4700-4706). In thepresent study, when Acolbifene was given to OVX animals, the mosttypical morphological feature induced by the SERM was the appearance ofa superficial layer of well-aligned small mucus cells overlying a basalcell layer, a pattern which was slightly more pronounced in thePremarin+Acolbifene group and which remained preponderant in all groupstreated with Acolbifene, including when the SERM was added to DHEA.Vaginal epithelium mucification under treatment with an antiestrogen hasbeen reported in immature (Anderson and Kang, 1975, Am J anat, 144(2):197-207) and adult (Yoshida et al., 1998, Cancer Lett, 134(1): 43-51)rats. Although this morphological pattern has been compared to theprogesterone-induced mucification (Anderson and Kang, 1975, Am J anat,144(2): 197-207), the molecular mechanism by which an antiestrogeninduces epithelial mucification remains unknown.

Many beneficial effects of DHEA have been reported in postmenopausalwomen (Labrie et al., 1997, J. Clin. Endocrinol. Metab., 82: 3498-3505;Labrie et al., 1991, Mol Cell Endocrinol, 78: C113-C118). Since nospecific receptor for DHEA has been characterized, the morphologicalchanges observed in the rat vagina after DHEA treatment reflect itsintracrine conversion into active sex steroids having androgenic and/orestrogenic action through intracrine mechanisms (Labrie et al., 1991,Mol Cell Endocrinol, 78: C113-C118). Those changes comprise intenseepithelial mucification, high compactness of delicate, finely wovenlamina propria collagen fibers and moderate muscularis thicknessincrease when compared to OVX animals. The two first morphologicalchanges are typical of androgenic effects while the third shows anestrogen-like activity, which is further supported by a concomitantincrease in progesterone receptor expression in the muscularis layer.

Since DHEA is transformed into either or both androgens and estrogens inperipheral tissues, the thick mucified multilayered epithelium observedin the present study after treatment of OVX animals with DHEA, suggestsa predominant androgenic effect of mucification in the rat vagina, aneffect which could mask any potential coexisting minor estrogenic effectat the epithelial level. A previous study has shown the samemucification effect in the rat vagina (Sourla et al., 1998, J SteroidBiochem. Mol. Biol., 66(3): 137-149); In that previous study, theintravaginal application of DHEA achieved a significant effect at a doseten times lower than that found to be active following application ofDHEA on the dorsal skin.

The present data indicate co-existing major androgenic and minorestrogenic actions of DHEA in the rat vagina. Moreover, the presentresults are well supported by the observation of the vaginal epitheliumof OVX animals which received the same topical application of DHEA thanthat used in our study, plus a subcutaneous dose of the antiandrogenFlutamide (FLU) (unpublished results). Indeed, the androgenic effect ofmucification produced by the androgenic component of DHEA was completelyreversed by FLU and resulted in a stratified squamous epithelium,typical of an estrogenic effect (data not shown). If DHEA would have anexclusive androgenic effect (mucification) in the rat vagina, thiseffect would be reversed by FLU and an atrophic epithelium similar tothat of OVX animals should then be observed. Previous studies ingonadectomized male and female rats have clearly established thattreatment with DHEA leads to stimulatory androgenic and/or estrogeniceffects on the prostate, seminal vesicle and uterus, depending upon thetarget tissue under investigation (Labrie et al., 1988, Endocrinol, 123:1412-1417).

The combination of DHEA+Acolbifene, on the other hand, shows a reductionin the extent of epithelial mucification, thus displaying the alternanceof a well-aligned mucus cell layer—an Acolbifene effect—andinvaginations of multilayered hypertrophied mucous cells, whichcorrespond to the androgenic effect of DHEA. Accordingly, Acolbifeneinhibits the partial estrogenic effect of DHEA, while the majorandrogenic counterpart of DHEA is maintained and a small estrogen-likeeffect of Acolbifene is added, the latter being best seen at the ostiumlevel. The vaginal weight decrease observed after the addition ofAcolbifene to DHEA is smaller than that obtained after addition ofAcolbifene to Premarin, thus illustrating the smaller estrogeniccomponent of DHEA which is reversed by the antiestrogen, while the majorandrogenic component is not affected. In addition, the increases inlamina propria thickness and in compactness of the collagen fibers, whencompared to those of OVX and intact animals respectively, in theDHEA+Acolbifene group, suggest, again, a potential role of androgens inincreasing collagen compactness and thickness, since the estrogeniccomponent of DHEA is blocked by Acolbifene.

Other studies have demonstrated that the action of DHEA in the ratmammary gland (Sourla et al., 1998, Endocrinol, 139: 753-764), skinsebaceous glands (Sourla et al., 2000, J Endocrinol, 166(2): 455-462)and bone mineral density (Martel et al., 1998, J Endocrinol, 157:433-442) is almost exclusively androgenic. Nevertheless, the presence ofan estrogenic action of DHEA in the rat vagina has been previouslydemonstrated through induction of vaginal opening and precociousovulation in immature rats treated with this compound, while DHT, anandrogen not aromatizable to estrogens, did not produce such effects(Knudsen and Mahesh, 1975, Endocrinol, 97(2): 458-468). The capacity ofrat vaginal tissue to aromatize androgens, especially Testo, is likelyto account for the major part of the estrogenic effect of DHEA in thisorgan (Lephart et al., 1989, Biol Reprod, 40(2): 259-267).Androst-5-ene-3β,17β-diol (5-diol), a DHEA metabolite known to bind theestrogen receptor (Shao et al., 1950, J Biol Chem, 250: 3095-3100;Poortman et al., 1975, J Clin Endocrinol Metab, 40(3): 373-379; VanDoorn et al., 1981, Endocrinol, 108: 1587-1594; Adams et al., 1981,Cancer Res, 41: 4720-4726), could also contribute to the estrogeniceffect (Poulin and Labrie, 1986, Cancer Res, 46: 4933-4937). Theproposed combination of the antiestrogen Acolbifene with DHEA would thusprevent any unwanted stimulatory effect of 5-diol. On the other hand,acolbifene would show additional benefits by preventing bone loss(Martel et al., 2000, J Steroid Biochem Mol Biol, 74 (1-2): 45-56).

To the best of our knowledge, no previous study has shown thestimulatory effects of DHEA on the compactness and morphology of thelamina propria's collagen fibers and, to a lesser extent, on themuscularis. Such actions of DHEA-derived androgens and estrogens couldhave beneficial effects on vaginal function in postmenopausal women andshould provide the substrates required for the action of inhibitors oftype 5 phosphodiesterase such as sildenafil or tadalafil, possibly viaandrogen or estrogen-induced endothelial nitric oxide synthase(eNOS)-mediated facilitation of vaginal smooth muscle relaxation(reviewed in Munarriz et al., 2003, J Urol, 170 (2 Pt 2): S40-S44,Discussion S44-S45). In fact, Acolbifene has been found to induce eNOSin human and rat endothelial cells (Simoncini et al., 2002, Endocrinol,143(6): 2052-2061).

The results obtained in all the groups involving DHEA treatment revealthat AR expression is up-regulated by the androgens derived from theintracrine transformation of DHEA in the rat vagina, in a similarfashion in the three tissue compartments.

The beneficial morphological changes, observed concomitantly with thestrong modulation of rat vaginal AR by androgens, suggest that decreasedserum levels of DHEA-derived androgens in postmenopausal women couldcontribute to the decreased vaginal health and eventually to the loss oflibido and sexual enjoyment observed in this age group. Decreased serumtotal Testo, free Testo and DHEA-S were indeed found in women whoconsulted for decreased sexual desire (Guay and Jacobson, 2002, J SexMarital Ther, 28 Suppl 1: 129-142).

In the present study, we have shown that treatment of OVX female ratswith DHEA or the combination of DHEA with Acolbifene and of DHEA withAcolbifene and Premarin reversed the OVX-induced atrophic changes foundin the vagina through a predominant androgenic effect, via AR, which wasreflected by intense epithelial mucification and increased compactnessof the collagen fibers in the lamina propria. Treatment with DHEA alonealso moderately increased muscularis thickness. Such data underline theimportance of sex steroids synthesized locally from DHEA and DHEA-S inperipheral target tissues. In addition to these beneficial effects,Acolbifene reduced the inflammation incidence, possibly through anestrogen-like induction of a protective keratinized squamous epitheliumat the ostium level and an antiestrogenic effect of mucification at theinternal level.

Various attempts have been made to solve the problem of vaginal dryness,often linked to dyspareunia and loss of sexual enjoyment. As an example,local vaginal estrogen preparations are often prescribed to providerelief but the endometrium may be stimulated by the unopposed estrogen(Mattson et al., 1989, Maturitas, 11: 217-222). In hysterectomizedpostmenopausal women, ERT is still used but with the well-knownincreased risk of developing breast cancer. Moreover, ERT decreasesserum androgen levels by increasing sex hormone binding globulin, whichmay induce a relative ovarian and adrenal androgen deficiency, thuscreating an additional rationale for concurrent physiologic androgenreplacement (Casson et al., 1997, Obstet Gynecol, 90(6): 995-998).

Example 2

Methods

Animals and Treatment

Ten to twelve week-old female Sprague-Dawley rats (Crl:CD(SD)Br)(Charles River Laboratory, St-Constant, Canada) weighing 215-265 g attime of ovariectomy were used. The animals were housed individually inan environmentally-controlled room (temperature: 22±3° C.; humidity:50±20%; 12-h light-12-h dark cycles, lights on at 07:15h). The animalswere allowed free access to tap water and a certified rodent feed (LabDiet 5002 (pellet), Ralston Purina, St-Louis, Mo.). The experiment wasconducted in an animal facility approved by the Canadian Council onAnimal Care (CCAC) and the Association for Assessment and Accreditationof Laboratory Animal Care (AAALAC) in accordance with the CCAC Guide forCare and Use of Experimental Animals.

Different experiments were performed: two 20-day and two 8-monthstudies. In each of the two first experiments, the animals were randomlydistributed between 3 groups of 13 or 14 animals as follows: 1) Intactcontrol; 2) Ovariectomized (OVX) control; 3) OVX+raloxifene (0.5 mg/kg),or, 3) OVX+LY 363381 (0.5 mg/kg). In one 8-month study, the animals wererandomly distributed between 5 groups of 7-8 animals as follows: 1)Intact control; 2) Ovariectomized (OVX) control; 3) OVX+Acolbifene (0.5mg/kg); 4) OVX+ERA-923 (0.5 mg/kg); 5) OVX+TSE-424 (0.5 mg/kg). In theother 8-month study, the animals were randomly distributed between 3groups of 10-11 animals as follows: 1) Intact control; 2) Ovariectomized(OVX) control; 3) OVX+lasofoxifene (0.5 mg/kg). On the first day of thestudy, the animals of the appropriate groups were bilaterallyovariectomized (OVX) under isoflurane anesthesia. The tested compoundswere then given once daily by oral gavage as a suspension in 0.4%methylcellulose (0.5 ml/rat) from day 1 to day 20 of the study or fromday 1 to week 32 of the study.

Twenty-four hours after the last dosing, the animals were sacrificedunder isoflurane anesthesia by exsanguination. The vaginae werecollected and immersed in 10% neutral buffered formalin immediatelyafter resection. The vaginae were then either cut in 7 cross-segments(for Acolbifene, ERA-923 and TSE-424) or cut to sample only the middlecross-segment. (raloxifene, lasofoxifene or LY 363381). The segmentswere then routinely processed and embedded in paraffin blocks. Whenappropriate, all the segments were positioned in the paraffin block in asequence corresponding to their original anatomical position andoriented perpendicular to the surface of the block, thus allowing thesegments to be cut in cross-sections. For each animal, a 4 μm-thickparaffin section was cut and stained with haematoxylin-eosin formorphological examination.

In FIGS. 10 and 11 is shown a comparison between different SERMs:vaginal epithelium morphology, in the OVX rat, at magnification 200×.Pictures A to D show the epithelial morphology of the first segment ofthe vagina (ostium). While the epithelium of the OVX animals is thin(4-6 cell layers), atrophic and does not display a visible granularlayer in A, the same epithelium is thicker in the 3 SERMs (8-11 celllayers) and displays a multilayered granular compartment, in B,C and D.In E to K, a segment situated in the middle of the vagina isillustrated. In the OVX animals, the vaginal epithelium is thin andatrophic (2-3 cell layers), in E, while the thickness is increased inall the treated groups (F to K), due to the presence of one (Acolbifene,ERA-923 and TSE-424) or many (raloxifene, lasofoxifene or LY 363381)mucous cell layer(s) overlying the basal cell layer.

Example 3

Materials and Methods

Ten to twelve week-old female Sprague-Dawley rats (Crl:CD®(SD)BrVAF/Plus™) (Charles River Laboratory, St-Constant, Canada) weighingapproximately 250-275 g at start of the experiment were used. Theanimals were acclimatized to the environmental conditions (temperature:22±3° C.; humidity: 50±20%; 12-h light-12-h dark cycles, lights on at07:15 h) for at least one week before starting the experiment. Theanimals were housed individually and were allowed free access to waterand rodent food (Lab Diet 5002, Ralston Purina, St-Louis, Mo.). Theexperiment was conducted in accordance with the CCAC Guide for Care andUse of Experimental Animals in an animal facility approved by theCanadian Council on Animal Care (CCAC) and the Association forAssessment and Accreditation of Laboratory Animal Care (AAALAC).

A total of 25 female rats were randomly distributed into 5 groups asfollows: 1) Three ovariectomized untouched control (OVX); 2) FourOVX+placebo suppository; 3) Six OVX+0.33 mg DHEA suppository; 4) SixOVX+0.66 mg DHEA suppository; 5) Six OVX+1.00 mg DHEA suppository. Onthe first day of the study, the animals of all groups were bilaterallyovariectomized (OVX) under isoflurane-induced anesthesia. The insertioninto the vagina of one suppository began on day 5 of the study and wascontinued once daily until day 18 of the study, for a total of 14 daysof treatment. The suppository was gently placed in the vaginal openingand then delicately pushed about 2-3 mm inside the vagina with the helpof a small, smooth glass stick, disinfected in 70% alcohol and driedbetween each animal.

Twenty-four hours after the last dosing, the animals were sacrificedunder isoflurane anesthesia by exsanguination. Vagina—including theurethra—and uterus were collected, and then trimmed as described below.For further analysis, two pieces of inguinal mammary gland were sampled,as well as a square piece of about 2×2 cm of shaved dorsal skin, thelatter being put and gently flattened on a piece of carton.

Preparation of the Suppositories

DHEA suppositories were prepared using Whitepsol H-15 base (Medisca,Montreal, Canada). Any other lipophilic base such as Fattibase, Wecobee,cocoa butter, theobroma oil or other combinations of Whitepsol basescould also be used. The proper amounts of DHEA were weighed to givefinal concentrations of 0.33 mg or 0.66 mg or 1.00 mg per suppository ofa final volume of 50 μl±3 μl. The proper amount of suppository basepellets to weight in order to obtain the final volume needed wascalculated based on the previously measured density of the suppositorybase, which was 47.5 mg per 50 μl. The suppository base was put in asmall beaker, melted in microwave at power 50% for about 3 min and 30sec and then transferred on a heating plate, which was adjusted at thelowest power in order to keep the suppository base at a temperature of50±3° C. The DHEA micronized powder (obtained from Scheweizerhall Inc.at a purity of 100%) was then added in the base, all at once, the wholeclump left to sink and to wet for 3 min before being meticulously andslowly crushed and dissolved with a smooth rounded-end glass rod.Finally, the base with the dissolved DHEA (the placebo was the basewithout DHEA) was poured, using a 1000 μl-Gilson pipette with apre-heated pipette tip, in tube racks containing a series of 50μl-moulds. These moulds were made from microcentrifuge tubes, each ofthem previously calibrated with 50 μl of water and cut at the level ofthe line drawn at the water surface. After 30 min of cooling at roomtemperature, the suppositories were put at 4° C. for another 30 min.Finally, the excess of base was trimmed with a razor blade and thesuppositories were removed from their moulds and stored in closed glassbottles at 4° C. until use. They were allowed to reach room temperaturebefore being applied to the animals.

Histological Procedures

The organs were immersed in 10% neutral buffered formalin immediatelyafter resection. Each vagina and uterus was divided into 8 equalcross-segments and 4 cross-segments including two from the cervix andtwo from each of the two proximal and distal horns, respectively. Thesegments were then routinely processed and embedded all together in thesame paraffin block. Within the paraffin block, all the segments werepositioned in a sequence corresponding to their original anatomicalposition and oriented perpendicular to the surface of the block, thusallowing the segments to be cut in cross-sections. For each animal, a 4μm-thick paraffin section was cut and stained with haematoxylin-eosinfor morphological examination.

Results

Morphology of the Epithelium

After examination of all vaginal segments, it was found that segments 4and 6 are representative to compare between treatments. Segment 4 wasthe most proximal to the ostium which did not display features of theexternal skin, and was not or to a lesser extent affected by the slightto moderate inflammatory changes usually found in the external area ofthe vaginal mucosa in control animals. Segment 6 represented the deepervaginal mucosa and is separated from the uterine cervix by only onesegment.

In general, the ventral side of the vagina displays a thicker and a moremucified epithelium when compared to the dorsal side.

Intact

As shown in FIG. 13, at estrus, which is under major estrogenicinfluence, the vaginal epithelium is thick and keratinized. It displaysfrom 12 to 18 cell layers in segment 4, and 12 to 15 layers in segment6. Contrary to the thick stratified squamous epithelium seen at theostium, the granular layers are not obvious in the internal vaginalepithelium. At metestrus, under minimal hormonal influence, the vaginalepithelium is thinner. It displays 6 to 10 cell layers in segment 4, and5 to 7 layers in segment 6.

Ovariectomized Untouched

As shown in FIG. 14, in segment 4, the vagina is lined by a squamousstratified epithelium of 2 to 6 cell layers. The thickness of thissegment is quite variable between animals. In segment 6, completeatrophy is observed, with 2 to 3 cell layers of flat cells. Some rarefoci of small cylindrical mucous cells could be seen throughout thewhole vagina of these animals. Of the three animals of this group, twodisplayed mild inflammatory changes in the external three segments.

Placebo

As shown in FIG. 15, in comparison with the OVX untouched group, thevaginal squamous stratified epithelium of the placebo group was slightlythicker, with segment 4 displaying 4 to 8 cell layers. However, insegment 6, the same morphology found in the OVX untouched group wasobserved, with 2-3 epithelial cell layers. As in OVX untouched animals,foci of small cylindrical mucous cells could be found throughout thevagina, slightly more when compared with the OVX untouched animals. Ofthe four animals of this group, three displayed mild inflammatorychanges in the external three segments.

DHEA 0.33 mg/Suppository

As shown in FIG. 16, in general, at low doses, the morphological effectof DHEA is characterized by well-aligned cylindrical mucous cells, whileat higher doses, the mucous cells enlarge (hypertrophy), vacuolate andpile up. In the present group, the effect of DHEA is slightlydiscernible in segment 2 and gradually increases in segments 3-4, wherethere is an alternance of well-aligned and hypertrophied mucous cells.The DHEA effect slowly decreases in segments 5 and 6 where almost onlywell-aligned mucous cells can be observed. The effect becomes verylittle in segment 7 and disappears in the lateral vaginal folds ofsegment 8 (which includes the beginning of the cervix). Thus, in segment4, the vaginal epithelium is stratified squamous with 3 to 7 celllayers. About 20 to 60% of the vaginal lining consists of well-alignedmucous cells in alternance with 15 to 50% hypertrophied mucous cells,overlying one or more squamous stratified epithelium layers, and withnon-mucified areas. In segment 6, the epithelial thickness is reduced toalmost the thickness found in OVX untouched animals. It consists of asquamous stratified epithelium of 2 to 5 cell layers, covered by a layerof well-aligned mucous cells. A large variation in the occurrence ofmucous cells was observed, these cells covering from 5 to 75% of thevaginal surface, in alternance with non-mucified areas. Of the sixtreated animals, four of them displayed mild inflammation, unevenlydistributed over the whole vagina.

DHEA 0.66 mg/Suppository

As shown in FIG. 17, in this group, the effect of DHEA is discernible insegment 2 and increases significatively in segments 3-4 and 5 where amajority of hypertrophied mucous cells are observed. The number ofhypertrophied mucous cells decreases in segments 6 and 7 where themajority of cells are well-aligned mucous cells. Thus, in segment 4, thestratified squamous epithelium is 3 to 6 cell layer-thick. About 5 to20% of the vaginal lining consists of well-aligned mucous cells inalternance with 10 to 75% of hypertrophied mucous cells, overlying oneor more squamous stratified epithelium layers. Non-mucified epithelialareas are also present. In segment 6, the epithelial thickness isreduced and consists of a squamous stratified epithelium of 2 to 4 celllayers, surmounted by a layer of well-aligned mucous cells. The amountof mucified areas varied from 20 to 80% of the vaginal surface, inalternance with non-mucified areas. Of the six treated animals, twodisplayed mild inflammation in the three external segments.

DHEA 1.00 mg/Suppository

As shown in FIG. 18, In this group, the effect of DHEA is alsodiscernible in segment 2, but it increases significatively in segments3-4 and 5 where the vast majority of hypertrophied mucous cells areobserved, and very slowly decreases in segments 6-7 and 8, where thevast majority of well-aligned mucous cells are observed. Thus, insegment 4, the stratified squamous epithelium displays 3 to 9 celllayers. Hypertrophied mucous cells overlying 3 to 4 squamous cell layerscover 50 to 70% of the vaginal surface, while well-aligned mucous cellscover 5 to 30% of the vaginal surface. Non-mucified areas areinterspersed between mucified cells. In segment 6, the epithelialthickness is also reduced and consists of 2 to 4 cell layers.Hypertrophied mucous cells overlying 1 to 3 squamous cell layers coverabout 30 to 100% of the vaginal surface while well-aligned mucous cellscover 0 to 70%. Few interspersed non-mucified areas are also observed.Of the six treated animals, four of them displayed mild inflammation inthe four external segments.

CONCLUSIONS

-   1. The vehicle used for rat intravaginal treatment has desirable    characteristics:    -   a. Easily prepared in calibrated mould;    -   b. Excellent capability to completely dissolve the DHEA        micronized powder, thus assuring a standardized dosage;    -   c. No significant tissue reaction and morphological alterations        of the vaginal mucosa: only a slight thickness increase of the        epithelium was observed in the first 3 to 4 segments, possibly        due to the manipulations accompanying the insertion of the        suppository.-   2. DHEA produced an effect on the vaginal epithelial morphology at    the lowest tested dose (0.33 mg/suppository). The effect is maximal    from the 3^(rd) to the 5^(th) segment, then decreases gradually to    disappear at the level of the 7^(th) segment.-   3. At the DHEA dose of 0.66 mg/suppository, the effect is also    present at the 3^(rd) segment and remains quite strong in the 6^(th)    segment.-   4. At the DHEA dose of 1.00 mg/suppository, the effect is strong    from segment 2-3 to the deep vaginal folds neighbouring the uterine    cervix.

PHARMACEUTICAL COMPOSITION EXAMPLES

Set forth below, by way of example and not of limitation, are severalpharmaceutical compositions utilizing preferred active SERM Acolbifene,preferred active sex steroid precursor DHEA, preferred estrogen17β-estradiol or premarin, preferred type 5 cGMP phosphodiesteraseinhibitor Sildenafil or Tadalafil. The concentration of activeingredient may be varied over a wide range as discussed herein. Theamounts and types of other ingredients that may be included are wellknown in the art.

Example A Tablet

Weight % Ingredient (by weight of total composition) DHEA 15.0 Gelatin5.0 Lactose 63.5 Starch 16.5

Example B Gelatin Capsule

Weight % Ingredient (by weight of total composition) DHEA 15.0 Lactosehydrous 70.0 Starch 4.8 Cellulose microcrystalline 9.8 Magnesiumstearate 0.4

Example C Tablet

Weight % Ingredient (by weight of total composition) Acolbifene 5.0 DHEA15.0 Gelatin 5.0 Lactose 58.5 Starch 16.5

Example D Gelatin capsule

Weight % Ingredient (by weight of total composition) Acolbifene 5.0 DHEA15.0 Lactose hydrous 65.0 Starch 4.8 Cellulose microcrystalline 9.8Magnesium stearate 0.4

Example E Tablet

Weight % Ingredient (by weight of total composition) Acolbifene 5.0 DHEA15.0 Premarin 0.5 Gelatin 5.0 Lactose 63.0 Starch 16.5

Example F Gelatin Capsule

Weight % Ingredient (by weight of total composition) Acolbifene 5.0 DHEA15.0 Premarin 0.5 Lactose hydrous 64.5 Starch 4.8 Cellulosemicrocrystalline 9.8 Magnesium stearate 0.4

Example G Tablet

Weight % Ingredient (by weight of total composition) Sildenafil 15.0DHEA 15.0 Gelatin 5.0 Lactose 48.5 Starch 16.5

Example H Gelatin Capsule

Weight % Ingredient (by weight of total composition) Sildenafil 15.0Acolbifene 5.0 DHEA 15.0 Lactose hydrous 50.0 Starch 4.8 Cellulosemicrocrystalline 9.8 Magnesium stearate 0.4

Example I Tablet

Weight % Ingredient (by weight of total composition) Acolbifene 5.0 DHEA15.0 Sildenafil 15.0 Gelatin 5.0 Lactose 43.5 Starch 16.5

Example J Gelatin Capsule

Weight % Ingredient (by weight of total composition) Acolbifene 5.0 DHEA15.0 Sildenafil 15.0 Lactose hydrous 50.0 Starch 4.8 Cellulosemicrocrystalline 9.8 Magnesium stearate 0.4

Example K Vaginal Cream

Weight % Ingredient (by weight of total composition) DHEA 1.0 acolbifene0.2 Emulsifying Wax, NF 18.0 Light mineral oil, NF 12.0 Benzyl alcohol1.0 Ethanol 95% USP 33.8 Purifed water, USP 34.0

Example L Vaginal Suppository

Weight % Ingredient (by weight of total composition) DHEA 0.66 to 2.0Whitepsol H-15 base 98 to 99.34DHEA suppositories were prepared using Whitepsol H-15 base (Medisca,Montreal, Canada). Any other lipophilic base such as Fattibase, Wecobee,cocoa butter, theobroma oil or other combinations of Whitepsol basescould used.

KIT EXAMPLES

Set forth below, by way of example and not of limitation, are severalkits utilizing preferred active SREM Acolbifene, preferred active a sexsteroid precursor DHEA, preferred estrogens 17β-estradiol or conjugatedestrogens, preferred type 5 cGMP phosphodiesterase inhibitor Sildenafilor Tadalafil. The concentration of active ingredient may be varied overa wide range as discussed herein. The amounts and types of otheringredients that may be included are well known in the art.

Example A

The SERM, estrogens and type 5 cGMP phosphodiesterase inhibitor areorally administered while the sex steroid precursor is applied locallyor percutaneously administered.

SERM Composition for Oral Administration (Capsules)

Weight % Ingredient (by weight of total composition) Acolbifene 5.0Lactose hydrous 80.0 Starch 4.8 Cellulose microcrystalline 9.8 Magnesiumstearate 0.4

SERM+Estrogens Composition for Oral Administration (Capsules)

Weight % Ingredient (by weight of total composition) Acolbifene 5.0Premarin 0.5 Lactose hydrous 79.5 Starch 4.8 Cellulose microcrystalline9.8 Magnesium stearate 0.4

SERM+estrogens+type 5 cGMP phosphodiesterase inhibitor composition fororal administration (capsules) Weight % Ingredient (by weight of totalcomposition) Acolbifene 5.0 Premarin 0.5 Sildenafil 15.0 Lactose hydrous64.5 Starch 4.8 Cellulose microcrystalline 9.8 Magnesium stearate 0.4

Sex steroid precursor composition for topical administration (gel)Weight % Ingredient (by weight of total composition) DHEA 2.0Caprylic-capric Triglyceride 5.0 (Neobee M-5) Hexylene Glycol 15.0Transcutol (diethyleneglycol 5.0 monomethyl ether) Benzyl alcohol 2.0Cyclomethicone (Dow corning 345) 5.0 Ethanol (absolute) 64.0Hydroxypropylcellulose (1500 cps) 2.0 (KLUCEL)

Example B

The SERM and the sex steroid precursor are orally administeredNon-Steroidal Antiestrogen composition for oral administration(capsules) Weight % Ingredient (by weight of total composition)Acolbifene 5.0 Lactose hydrous 80.0 Starch 4.8 Cellulosemicrocrystalline 9.8 Magnesium stearate 0.4

Sex steroid precursor composition for oral administration

Gelatin Capsule

Weight % Ingredient (by weight of total composition) DHEA 15.0 Lactosehydrous 70.0 Starch 4.8 Cellulose microcrystalline 9.8 Magnesiumstearate 0.4

Other SERMs (Toremifene, Ospemifene, Raloxifene, Arzoxifene,Lasofoxifene, TSE-424, ERA-923, EM-800, SERM 3339, GW-5638) may besubstituted for Acolbifene in the above formulations, as well as othersex steroid inhibitors may be substituted for DHEA, other estrogens maybe substituted for Premarin and other type 5 cGMP phosphodiesteraseinhibitor may be substituted for Sildenafil or Tadalafil or byprostaglandin E1. More than one SERM or more than one precursor orestrogens or type 5 cGMP phosphodiesterase inhibitor may be included inwhich case the combined weight percentage is preferably that of theweight percentage for the single precursor or single SERM given in theexamples above.

The invention has been described in terms of preferred embodiments andexamples, but is not limited thereby. Those of skill in the art willreadily recognize the broader applicability and scope of the inventionwhich is limited only by the patent claims herein.

1. A method of treating or reducing the likelihood of acquiring problemsaffecting the layer lamina propria the and/or the layer muscularis ofthe vagina in postmenopausal women, said method comprising administeringa sex steroid precursor selected from the group consisting ofdehydroepiandrosterone, dehydroepiandrosterone sulfate,androst-5-ene-3b,17b-diol and 4-androsten-3,17-dione to a patient inneed of said treatment.
 2. The method of claim 1 further comprising thestep of administering to said patient a therapeutically effective amountof a selective estrogen receptor modulator as part of a combinationtherapy.
 3. The method of claim 1 further comprising the step ofadministering to said patient a therapeutically effective amount of anestrogen as part of a combination therapy.
 4. A method of treating orreducing the likelihood of acquiring inflammation of the vagina inpostmenopausal women, said method comprising administering to saidpatient a therapeutically effective amount of a selective estrogenreceptor modulator.
 5. A method of increasing the number and size ofmucous cells in the vaginal epithelium in postmenopausal women, saidmethod comprising administering to said patient a therapeuticallyeffective amount of a selective estrogen receptor modulator.
 6. Themethod of claim 1 wherein vaginal diseases selected from the groupconsisting of vaginal dryness, dyspareunia, and sexual dysfunction aretreated, or the risk of acquiring such diseases is reduced.
 7. A methodof increasing of the collagen fibers in the lamina propria of thevaginal wall in postmenopausal women, said method comprisingadministering a sex steroid precursor selected from the group consistingof dehydroepiandrosterone, dehydroepiandrosterone sulfate,androst-5-ene-3b,17b-diol and 4-androsten-3,17-dione to a patient inneed of said treatment.
 8. A method of increasing the muscaralisthickness of the vaginal wall in postmenopausal women, said methodcomprising administering a sex steroid precursor selected from the groupconsisting of dehydroepiandrosterone, dehydroepiandrosterone sulfate,androst-5-ene-3b,17b-diol and 4-androsten-3,17-dione to a patient inneed of said treatment.
 9. A method of treating or reducing thelikelihood of acquiring sexual dysfunction in postmenopausal women, saidmethod comprising administering a therapeutically effective amountselective estrogen receptor modulator to said patient in need of saidtreatment and further comprising the step of administering to saidpatient a therapeutically effective amount a type 5 cGMPphosphodiesterase inhibitor or a prostaglandin as part of a combinationtherapy.
 10. The method of claim 9 futher comprising the step ofadministering to said patient a therapeutically effective amount of asex steroid precursor selected from the group consisting ofdehydroepiandrosterone, dehydroepiandrosterone sulfate,androst-5-ene-3b,17b-diol and 4-androsten-3,17-dione.
 11. The method ofclaim 1 wherein the sex steroid precursor is dehydroepiandrosterone. 12.The method of claim 1 wherein the sex steroid precursor isdehydroepiandrosterone sulfate.
 13. The method of claim 1 wherein thesex steroid precursor is androst-5-ene-3b,17b-diol.
 14. The method ofclaim 9 wherein the type 5 cGMP phosphodiesterase inhibitor is selectedfrom the group consisting of Sildenafil(1-[[3-(4,7-Dihydro-1-methyl-7-oxo-3-propyl-1Hpyrazolo[4,3-d]pyrimidin-5-yl)-4-ethoxyphenyl]sulfonyl]-4-methylpiperazine),Tadalafil(6R,12aR)-6-(1,3-Benzodioxol-5-yl)-2,3,6,7,12,12a-hexahydro-2-methylpyrazino[1′,2′:1,6]pyrido[3,4-b]indole-1,4-dione,DA-8159, EMR-62203, TA-1790, UK-371800 and SCH-446132.
 15. The method ofclaim 9 wherein the prostaglandin is Prostaglandin E1 (alprostadil;(11a,13E,15S)-11,15-Dihydroxy-9-oxoprost-13-en-1-oic acid))
 16. Themethod of claim 1 wherein the sex steroid precursor is administeredintravaginally.
 17. A pharmaceutical composition for vaginal applicationcomprising: a) a pharmaceutically acceptable excipient, diluent orcarrier; and b) a therapeutically effective amount of at least one sexsteroid precursor selected from the group consisting ofdehydroepiandrosterone, dehydroepiandrosterone-sulfate, andandrost-5-ene-3b,17b-diol.
 18. The pharmaceutical composition of claim17, additionally comprising a therapeutically effective amount of atleast one selective estrogen receptor modulator.
 19. The pharmaceuticalcomposition of claim 18, additionally comprising a therapeuticallyeffective amount of at least one estrogen.
 20. A pharmaceuticalcomposition comprising: a) a pharmaceutically acceptable excipient,diluent or carrier; b) a therapeutically effective amount of at leastone selective estrogen receptor modulator; and c) a therapeuticallyeffective amount of at least one the type 5 cGMP phosphodiesteraseinhibitor.
 21. A pharmaceutical composition comprising: a) apharmaceutically acceptable excipient, diluent or carrier; b) atherapeutically effective amount of at least one selective estrogenreceptor modulator; c) a therapeutically effective amount of at leastone the type 5 cGMP phosphodiesterase inhibitor; and d) atherapeutically effective amount of at least one sex steroid precursorselected from the group consisting of dehydroepiandrosterone,dehydroepiandrosterone-sulfate, androst-5-ene-3b,17b-diol.
 22. A kitcomprising a first container containing a therapeutically effectiveamount of at least one type 5 cGMP phosphodiesterase inhibitor selectedfrom the group consisting of Prostaglandin E1, Sildenafil and Tadalafil,and further comprising a second container containing a therapeuticallyeffective amount of at least one selective estrogen receptor modulator.23. A kit comprising a first container containing a therapeuticallyeffective amount of at least one sex steroid precursor selected from thegroup consisting of dehydroepiandrosterone,dehydroepiandrosterone-sulfate, androst-5-ene-3b,17b-diol, and furthercomprising a second container containing a therapeutically effectiveamount of at least one selective estrogen receptor modulator and a thirdcontainer containing a therapeutically effective amount of at least onetype 5 cGMP phosphodiesterase inhibitor or Prostaglandin E1.
 24. Themethod of claim 2 wherein the selective estrogen receptor modulator hasa molecular formula with the following features: a) two aromatic ringsspaced by 1 to 2 intervening carbon atoms, both aromatic rings beingeither unsubstituted or substituted by a hydroxyl group or a groupconverted in vivo to hydroxyl; and b) a side chain possessing anaromatic ring and a tertiary amine function or salt thereof.
 25. The kitof claim 22 wherein the selective estrogen receptor modulator has thefollowing formula:

wherein R₁ and R₂ are independently hydrogen, hydroxyl or a moiety whichis converted to hydroxyl in vivo; wherein Z is a bivalent closingmoiety; wherein the R100 is a bivalent moiety which distances L from theB-ring by 4-10 intervening atoms; wherein L is a bivalent or trivalentpolar moiety selected from the group of —SO—, —CON—, —N<, and —SON<;wherein G₁ is selected from the group consisting of hydrogen, a C₁ to C₅hydrocarbon or a bivalent moiety which joins G₂ and L to form a 5- to7-membered heterocyclic ring, and halo or unsaturated derivatives of theforegoing. wherein G₂ is either absent or selected from the groupconsisting of hydrogen, a C₁ to C₅ hydrocarbon or a bivalent moietywhich joins G₁ and L to form a 5- to 7-membered heterocyclic ring, andhalo or unsaturated derivatives of the foregoing. wherein G₃ is selectedfrom the group consisting of hydrogen, methyl and ethyl.
 26. The kit ofclaim 25, wherein the selective estrogen receptor modulator is anoptically active compound having an absolute configuration S on carbon 2or pharmaceutically acceptable salt thereof, said compound having themolecular structure:

wherein R₁ and R₂ are independently selected from the group consistingof hydroxyl and a moiety convertible in vivo to hydroxyl; wherein R³ isa species selected from the group consisting of saturated, unsaturatedor substituted pyrrolidinyl, saturated, unsaturated or substitutedpiperidino, saturated, unsaturated or substituted piperidinyl,saturated, unsaturated or substituted morpholino, nitrogen-containingcyclic moiety, nitrogen-containing polycyclic moiety, and NRaRb (Ra andRb being independently hydrogen, straight or branched C₁-C₆ alkyl,straight or branched C₂-C₆ alkenyl, and straight or branched C₂-C₆alkynyl).
 27. The method claim 24 wherein said compound or saltsubstantially lacks (2R)-enantiomer.
 28. The method of claim 24 whereinthe benzopyran derivative is a salt of an acid selected from the groupconsisting of acetic acid, adipic acid, benzenesulfonic acid, benzoicacid, camphorsulfonic acid, citric acid, fumaric acid, hydroiodic acid,hydrobromic acid, hydrochloric acid, hydrochlorothiazide acid,hydroxy-naphthoic acid, lactic acid, maleic acid, methanesulfonic acid,methylsulfuric acid, 1,5-naphthalenedisulfonic acid, nitric acid,palmitic acid, pivalic acid, phosphoric acid, propionic acid, succinicacid, sulfuric acid, tartaric acid, terephthalic acid, p-toluenesulfonicacid, and valeric acid.
 29. The method of claim 2 wherein said selectiveestrogen receptor modulator is:


30. The method of claim 24 wherein the compound is selected from thegroup consisting of:(−)-cis-(5R,6S)-6-phenyl-5-[4-(2-pyrrolidin-1-ylethoxy)phenyl]-5,6,7,8-tetrahydronaphthalen-2-ol,D-(−)-tartrate salt (lasofoxifene).
 31. The method of claim 1 whereinthe sex steroid precursor is dehydroepiandrosterone.